Mutation detection by competitive oligonucleotide priming

ABSTRACT

The present invention relates to a process for the rapid and simple detection of mutations in DNA and differences between DNA sequences. This competitive oligonucleotide priming system can be used for the detection of any differences between DNA sequences for which a DNA sequence is known.

This is a continuation of application Ser. No. 170,214, filed Mar. 18,1988 , abandoned.

FIELD OF THE INVENTION

This invention relates to the field of detecting differences (mutations)in genetic sequences by competitive oligonucleotide priming. The methodof detection is useful in a variety of areas including screening forgenetic disease, infectious disease, and cancer; forensic medicine;animal husbandry including breeding for agriculture and recreationalpurposes.

BACKGROUND

This invention is an improvement on currently established procedures forthe detection of differences in DNA sequences. The detection ofdifferences in DNA sequences is a desirable and necessary procedure inthe following exemplary areas; detection and diagnosis of allelesresponsible for genetic diseases in humans and other species; detectionand diagnosis of DNA sequences associated or linked to genes that may ormay not be involved in disease in humans and other species; detectionand diagnosis of neoplasms and the effects of therapy on neoplasms;detection of and distinction between different pathogens (e.g., viruses,bacteria and fungi); determining the purity of animal strains andpedigrees; distinguishing and identifying different human and animalsamples in forensic medicine.

Frequently the DNA sequence difference to be detected is a single DNAbase substitution (point mutation). DNA is normally composed of variouscombinations of four bases termed Adenine (A); Thymidine (T); Cytosine(C) and Guanosine (G). Thus, an example of a DNA sequence may beATCGCGATCGT. A point mutation may be the substitution of any of thethree bases not normally found at a single position for a base that isnormally found at that position. For example, the transmutation of a DNAsequence ATCG CGATCGT to ATCG GGATCGT is a point mutation at theunderlined position.

Although point mutations may not account for the majority of differencesbetween randomly selected DNA sequences, they do account for manydifferences between DNA sequences that are responsible for polymorphismsand "disease" related DNAs.

DNAs that differ only by point mutation are very difficult todistinguish by current technologies. Procedures for detecting pointmutations fall into two main categories: (1) procedures which detectpoint mutations when the precise DNA sequence change can be anticipated;(2) procedures which "scan" for point mutations where the precise natureof the individual DNA gene change is not known. The present inventionwill work in either situation.

Prior to the present invention, point mutations where there is someknowledge of the DNA sequences differences between the normal andvariant DNA have been detected by:

(1) Restriction fragment length polymorphisms (D. Botstein, et al. Am.J. Hum. Genet., 32:314-331 (1980)) or (2) Allele specificoligonucleotide (ASO) probing (G. Angelini, P.N.A.S. (U.S.A.),83:4489-4493 (1986).

In the restriction fragment length polymorphism procedure, restrictionendonucleases are used to cut the DNA into various chain lengths whichcan be measured. In allele specific oligonucleotide probing, single basemismatches are determined by thermodynamic differences. The annealingconditions are set such that perfectly paired strands anneal andnon-perfectly paired strands do not anneal.

The polymerase chain reaction (PCR) exemplified by U.S. Pat. Nos.4,683,202 and 4,683,195 is used to amplify specific DNA sequences,however, PCR does not, by itself, provide a method to detect single basemutations. The PCR may be used in conjunction with other techniques suchas the present invention to detect point mutations and other DNAsequence differences.

The current invention, competitive oligonucleotide priming (COP),distinguishes closely related DNA sequences by comparing competitiveannealing of two or more DNA sequences closely matched to the DNAsequence of interest. The COP procedure has some similarity to theAllele specific oligonucleotide probing procedure and to the polymerasechain reaction procedure, however, neither ASO probing or PCRamplification procedures utilize the unique competitive annealing assayof the present invention to detect specific sequences differing by asingle base.

The COP procedure of the present invention has the advantages ofsimplicity and speed. Furthermore, no filter for hybridization isneeded, it can be used with solid supports and the whole procedure canbe automated for decreased cost. It provides a method to solve a longfelt need to improve and simplify the detection of single base changesin DNA sequences.

SUMMARY OF THE INVENTION

An object of the present invention is a method for detecting a specificknown polynucleotide sequence.

An additional object of the present invention is a method fordistinguishing between different nucleotide sequences.

A further object of the present invention is the detection of geneticdisease.

An additional object of the present invention is a method to detectgenetic polymorphism in known genetic sequences.

Thus, in accomplishing the foregoing objects there is provided inaccordance with one aspect of the present invention a method fordetecting the presence or absence of a specific known nucleic acidsequence or distinguishing between different sequences comprising thesteps of:

adding competitive oligonucleotide primers to a sample of nucleic acidor mixture of nucleic acids, wherein said competitive oligonucleotideprimers include at least two primers, one being substantiallycomplementary to the specific known sequence and at least one having abase mismatch with the specific known sequence;

preferentially hybridizing the substantially complementary primer to thespecific known sequence under competitive conditions;

extending the preferentially hybridized primer from its 3' terminus tosynthesize an extension product complementary to the strand to which theprimer is hybridized; and

identifying said extension product.

Additional embodiments of the invention include attaching labels to thecompetitive oligonucleotide primers for the easy detection of theextension products. These labels include radioisotopes, fluorescers,chemiluminescer, enzymes and antibodies.

In other embodiments, the sequence to be detected is amplified duringthe competitive oligonucleotide primer assay and/or prior to performingthe competitive oligonucleotide primer assay.

A further embodiment employs multiple competitive oligonucleotideprimers each labeled differently to simultaneously detect geneticpolymorphism at a single locus or to detect different loci.

Another embodiment includes using the competitive oligonucleotide primermethod in detecting genetic disease, forensic medicine, paternitytesting, gene mapping, pathogen detection and neoplasia screening andtherapy.

An additional embodiment includes dividing a sample of nucleic acid froman individual and performing competitive oligonucleotide primer assayssimultaneously on the different portions.

The present invention is useful in detecting genetic polymorphisms.Specific applications include: detecting genetic diseases such as sicklecell anemia, α₁ -antitrypsin deficiency and hemophilia; screening fordisease association by linkage analysis; tissue typing; gene mapping;screening for neoplasms and the effect of therapy; detection of knownpathogens, (for example viruses bacteria, yeast and fungi); determiningthe pedigrees and/or purity of animal strains; and disease screening inanimals.

Other and further objects features and advantages will be apparent fromthe following description of the presently preferred embodiments of theinvention given for the purpose of disclosure when taken in conjunctionwith the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more readily understood from a reading of thefollowing specification and by references to the accompanying drawings,forming a part thereof:

FIG. 1A-C demonstrate the basic principle of the competitiveoligonucleotide priming system;

FIG. 1A demonstrates that when two closely related primers compete for asingle DNA template, a perfectly matched primer will anneal or hybridizewith the template in preference to a primer with a single base mismatch;

FIG. 1B demonstrates that an oligonucleotide primer can be maderadioactive to facilitate its detection.

FIG. 1C the demonstrates detection of competitive oligonucleotidepriming by the polymerase chain reaction (PCR). A DNA template is primedat two sites. A single oligonucleotide primer is used at one of thesites (common primer) and the competing oligonucleotide primers are usedat the other site, which includes the DNA mutation. After PCRamplification, the `correct`, perfectly matched primer is incorporatedinto the PCR amplification product.

FIG. 2A-B shows the DNA sequence of regions surrounding theoligonucleotide priming sites in Examples 1 and 2.

FIG. 2A shows the M13mp18 filamentous phage DNA sequence.

FIG. 2B shows the ornithine transcarbamylase cDNA sequence.

FIG. 3A demonstrates the analysis of competitive oligonucleotide primingproducts generated from primers specific to + and spf OTC alleles.

FIG. 3B shows autoradiographs of the gel shown in 3A demonstrating thatthe radioactive primers have been incorporated into a 72 nucleotidefragment in lanes 1 and 4, where the radioactive primers perfectly matchthe template, while the radioactive mismatch primers in lanes 2 and 3have been excluded.

FIG. 4A demonstrates the autoradiographic analysis of fragmentsgenerated from COP and PCR of M13mp18 DNA using 12-mer oligonucleotidesin the competition.

FIG. 4B demonstrates the autoradiographic analysis of fragmentsgenerated from COP and PCR of M13mp18 DNA using 20-mer oligonucleotidesin the competition. The drawings are not necessarily to scale andcertain features of the invention may be exaggerated in scale or shownin schematic form in the interest of clarity and conciseness.

DETAILED DESCRIPTION

It will be readily apparent to one skilled in the art that varioussubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

The term "oligonucleotide primers" as used herein defines a moleculecomprised of more than three deoxyribonucleotides or ribonucleotides.Its exact length will depend on many factors relating to the ultimatefunction or use of the oligonucleotide primer including temperature,source of the primer and use of the method. The oligonucleotide primercan occur naturally as in a purified restriction digest or be producedsynthetically. The oligonucleotide primer is capable of acting as apoint of initiation of synthesis when placed under conditions whichinduce synthesis of a primer extension product complementary to anucleic acid strand. The conditions can include the presence ofnucleotides and an inducing agent such as DNA polymerase at a suitabletemperature and pH. Although the primer preferably is single stranded,it may alternatively be double stranded. If it is double stranded, theprimer must first be treated to separate its strands before it is usedto produce extension products. In the preferred embodiment, the primeris an oligodeoxyribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of theinducing agent. In the competitive oligonucleotide primer method,oligonucleotides can range from about 8 to 30 mers in length. In thepreferred embodiment, the competitive primers are 12 to 16 mers inlength. The sensitivity and specificity of the competitiveoligonucleotide primer assay is determined by the length. Primers whichare too short, i.e., less than about 8 mers show non-specific binding toa wide variety of sequences in the genome and thus are not very useful.On the other hand, primers which are too long, i.e., greater than about30 mers do not show competitive binding because a single base mismatchusually does not affect the binding efficiency in long oligonucleotides.

As used herein "competitive oligonucleotide primers" shall refer tothose oligonucleotide primers which differ by at least one basemismatch. This difference or differences results in a differential rateand ability to bind to the known nucleotide sequence. By controlling therate and ability to bind the competitive oligonucleotide primer can beadvantageously used. A variety of conditions including temperature,ionic strength and the chemical composition of the buffer will alter thebinding capacity. Under appropriate conditions, when competitiveoligonucleotide primers are incubated with a DNA template, theoligonucleotide sequence which most nearly matches the known sequence tobe hybridized will bind preferentially over the sequence which has abase mismatch or the most base mismatches.

As used herein "base mismatch" shall refer to a change in thenucleotides, such that when a primer lines up with the known sequence anabnormal bonding pair of nucleotides is formed. Normally guanine (G) andcytosine (C) bind and adenine (A) and thymine (T) bind in the formationof double stranded nucleic acids. Thus, the standard base pairing, A-Tor G-C, is not seen in base mismatched pairing. A variety of basemismatches can occur, for example G-G, C-C, A-A, T-T, A-G, A-C, T-G, orT-C. This mispairing, and its effects on the efficiency of annealing isone basis for the competitive binding of the oligonucleotide primers.

As used herein a "common primer" is a primer which binds to the strandcomplementary to the strand that the competitive oligonucleotide primersbind and it binds at a site distant from the competitive oligonucleotideprimers. This distance should be sufficient to allow the synthesis ofextension product between the two binding sites, yet close enough suchthat the extension products of the common primer(s) overlap thecompetitive oligonucleotide primer(s) and the extension product of thecompetitive oligonucleotide primer(s) overlaps the common primer(s). Theextension products from the common primer(s) and competitive primer(s)are complementary to each other.

All the oligonucleotide primers used herein are selected to besubstantially complementary to the different strands (templates) of eachknown specific sequence to be detected so that the primers hybridizewith their respective strands. The primer sequence need not reflect theexact sequence of the template in the competitive oligonucleotide primerassay. However, it is important that the different sequences used ascompetitive oligonucleotide primers have different numbers of basemismatches. For example, in the detection of a normal genetic sequence,the competitive oligonucleotide primers could include a primer which isan exact copy of the complementary strand to the normal genetic sequenceand a primer which is a copy of the complementary strand with one basepair mismatched (see FIG. 1A). Both a perfectly matched primer and aprimer with a single DNA base mismatch are able to bind to the template.However, when the two closely related primers are incubated togetherwith the DNA template, the binding of the perfectly matched primer willbe favored over a primer with a single base mismatch. Alternatively, oneof the primers can contain one base mismatch to the known geneticsequence and the other oligonucleotides would contain at least twomismatches. Thus, generally the requirements are that one of thesequences have N mismatches and the other sequence or sequences havegreater than N mismatches, where N can be from zero to any number ofmismatches which will still provide a substantially similar sequenceable to bind. When two oligonucleotides differing by a single DNA baseare supplied as primers in a reaction containing a single DNA or RNAtemplate then the perfectly matched oligonucleotide primer will behighly favored over the primer with the single base mismatch. Similarly,if neither primer is a perfect match the more closely matched primerwill be favored. The greater the difference between the sequence ofinterest and the other sequences, the more efficiently the competitiveoligonucleotide primer assay functions. However, when the difference istoo great, it may no longer function as a competitive assay.

As used herein the term "genetic polymorphism" refers to the variationseen at a genetic locus wherein two or more different nucleotidesequences can coexist at the same genetic locus in the DNA. Thedifferent sequences may or may not result in disease. For example, HL-Ahaplotypes have different genetic sequences which vary but do not resultin disease while sickle cell anemia is a disease caused by a singlechange in the genetic sequence.

As used herein the "normal genetic sequence" refers to that sequence,which in the case of genetic disease results in the normal phenotype, orin the case of no disease results in the most common haplotype found inthe population.

As used herein the "mutated genetic sequence" refers to that sequencewhich has at least one base change difference in the DNA sequence fromthe normal genetic sequence. The mutant sequence or sequences areresponsible for the genetic disease or the less common haplotypesexpressed at a given locus.

As used herein the term "extension product" shall be that product whichis synthesized from the 3' end of the oligonucleotide primer and whichis complementary to the strand to which the oligonucleotide primer isbound. A "competitive extension product" shall refer to the extensionproduct which is synthesized from the 3' end of one of the competitiveoligonucleotide primers.

As used herein term "differentially labeled" shall indicate that theeach competitive oligonucleotide primer has a different label attached.One skilled in the art will recognize that a variety of labels areavailable. For example, these can include radioisotopes, fluorescers,chemiluminescers, enzymes and antibodies. Various factors affect thechoice of label. These include the effect of the label on the rate ofhybridization and binding of the primer to the DNA, the sensitivity ofthe label, the ease of making the labeled primer, ability to automate,available instrumentation, convenience and the like. For example, in themethods employing differentially labeled primer, each primer could belabeled with a different radioisotope such as ³² P, ³ H and ¹⁴ C or eachprimer could be labeled with a different isotope of the same element; adifferent fluorescer such as fluorescin, tetramethylrhodamine, Texas redand 4-chloro-7-nitrobenzo -2-oxa-1-diazole (NBD); or a mixture ofdifferent labels such as radioisotopes, fluorescers andchemiluminescers. In these examples, each primer can be differentiatedfrom all other primers when they are in a mixture.

The specific known nucleic acid sequence which is being detected hereinmay be derived from any source(s) in purified or non-purified form.Sources can include plasmids and cloned DNAs, genomic DNA from anysource including bacteria, fungi, yeast, viruses and higher organismssuch as plants, birds, reptiles and mammals. In the preferredembodiment, the source is genomic DNA. The genomic DNA can be preparedfrom blood, urine, tissue material, such as chorionic villi and amnioticcells, by a variety of techniques known to one skilled in the art.

Any specific known nucleic acid sequence can be detected by the presentmethod. It is only necessary that a sufficient number of bases at bothends of the sequence be known in sufficient detail to prepare twooligonucleotide primers which will hybridize to the different strands ofthe desired sequence at relative positions along the sequence. Afterhybridization of the primers, an extension product is synthesized fromone primer. When the extension product is separated from its template,it can serve as a template for extension of the other primer into anucleic acid of defined length. The greater the knowledge about thebases at both ends of the sequence, the greater can be the specificityof the primers for the targeted nucleic acid sequence, and thus, thegreater the efficiency of the process.

The oligonucleotide primers may be prepared using any suitable method,for example, the phosphyltriester and phosphyldiester methods orautomated embodiments thereof, the synthesis of oligonucleotides on amodified solid support, the isolation from a biological source(restriction endonuclease digestion), and the generation byenzymatically directed copying of a DNA or RNA template.

One embodiment of the present invention is a method for detecting thepresence or absence of a specific known nucleic acid sequence, ordistinguishing between different sequences, comprising the steps of:adding competitive oligonucleotide primers to a sample of nucleic acidor a mixture of nucleic acids, wherein said competitive oligonucleotideprimers include at least two primers, one being substantiallycomplementary to the specific known sequence and at least one having abase mismatch with a specific known sequence; preferentially hybridizingthe substantially complementary primer to the specific known sequenceunder competitive conditions; extending the preferentially hybridizedprimer from its 3' terminus to synthesize an extension productcomplementary to the strand to which the primer is hybridized; andidentifying the extension product.

According to the present invention, two closely related DNA sequencesmay be distinguished by competitive oligonucleotide priming.

Basically, in order to detect a DNA sequence which differs by one ormore bases from a known DNA sequence, oligonucleotide primers whichmatch the known DNA sequence and oligonucleotide primers which differfrom the matched oligonucleotide primers by at least one base pair areincubated in the presence of the DNA to be tested. At least one of theprimers may be detectably labeled as described below. The assay of thepresent invention detects the preference for more closely matched DNAprimers to anneal or hybridize to its corresponding template bydetermining which of the labeled primers is incorporated into theextension product.

For example, the reaction conditions can include two oligonucleotideprimers, preferably in at least a molar excess of primer to template.The deoxyribonucleoside triphosphates dATP, dCTP, dGTP and TTP are addedin adequate amounts to provide sufficient substrate for the synthesis ofthe new DNA strands and at concentrations sufficient for the activity ofthe polymerase to be used. The resulting solution is heated to about100° C. for from about 15 seconds to about 2 minutes and preferablyabout 1 minute in order to denature any double stranded species. Avariety of buffers can be used to support the competitive hybridization.It is required that the stringency of the buffer conditions is such thatthe best matched primer is able to bind efficiently to allow DNAextension. The buffer must also allow the enzymes that catalyze the DNAextension to function. The amount of the primer that is present must begreater than the molar amount of the template that is present, howeverit is not necessary that each of the primers are present in the samemolar amount.

The length of the denaturation period may vary. It is only necessarythat it be sufficient to allow denaturation of any double strandedtemplate species in the mixture. The temperature at which thedenaturation is carried out may also vary depending upon the otherdenaturation conditions such as, buffer constituents, length ofdenaturation period, level or concentration of double strandedcomponents in the mixture, and physical characteristics, such as meltingtemperature, of the double stranded component(s). Preferably, thetemperature for the denaturation process ranges from about 90° C. to110° C.; most preferably, the denaturation is carried out at about 105°C.

After the denaturation is complete, the solution is allowed to cool andthe primers are allowed to hybridized (or anneal) to the templatestrands under competitive conditions. The annealing temperature may varyfrom about 10° C. to 65° C., preferably, 28° C. The ideal temperaturemay be determined according to methods known to those of skill in theart and will be dependent upon factors such as the melting temperatureof the best match primer, as well as the other assay conditionsdescribed above. The annealing process is allowed to proceed for atleast 5 seconds. Preferably, the annealing process is carried out at 28°C. for 30 seconds.

After the annealing process is complete, an inducing or catalyzing agentis introduced into the solution to start the primer extension reaction.The inducing or catalyzing agent may be any agent that promotes theextension of the oligonucleotide primer, such as the Klenow fragment ofDNA polymerase 1 from E. coli or the heat-stable DNA polymerase fromThermus aquaticus (Taq polymerase). The amount of catalyzing agent addedwill depend upon the inherent activity of the preparation and will beknown to one of skill in the art, for instance, when the E. coli Klenowfragment is used as the catalyzing agent, at least 0.1 to 100 Units. Oneunit of Klenow activity may be defined as that amount of enzyme thatwill catalyze the incorporation of 10 nM of total deoxyribonucleotidesinto acid precipitable material in 30 minutes at 37° C. usingpoly[d(A-T)] as template-primer. Preferably, 5 Units are added. Variousconditions are known to one skilled in the art, but the extensionreaction can occur at 8° C. to 90° C. using, for example, DNA polymerase(Klenow) or heat stable DNA polymerase (Taq). The extension reactionsare usually done in a final volume of 100 microliters containing 30 mMTris-acetate, pH 7.9, 60 mM sodium acetate, 10 mM magnesium acetate, 10mM dithiothreitol, 1.5 mM each of the dATP, TTP, dCTP and dGTP, 4 μM ofeach primer or primer family and about 0.5 to 1 μM of DNA.

Depending on the method used to identify the extension product, thesteps involved will vary. For example, if the common primer is attachedto a solid support, the sequences of the extension primer binding to theknown sequence will be bound to the solid support. Thus, detecting thepresence or absence of a sequence on the solid support will allowidentification of the primer. On the other hand, if the primers are notattached to a solid support, it may be necessary to treat the doublestranded extension product-template to form single strands. One skilledin the art will recognize that physical, enzymatic and chemical meansare available to separate the strands. Typically heat denaturation isused.

A variety of methods are known in the art for the detection of nucleicacid sequences. For example, nucleic acid sequences can be labeled withradioisotopes, fluorescers, chemiluminescers, enzymes and antibodies.The presence or absence of the label indicates whether the extensionproduct is from that specific primer (FIG. 1B). Alternatively, thesequence of interest could contain a restriction endonuclease site whichis different in the normal and mutated sequences. In this case thedouble stranded extension product-template would not be separated, butrather would be submitted to restriction endonuclease digestion and theresultant restriction fragment lengths measured.

Another embodiment of the method includes the further step of amplifyingthe extension products prior to the identifying step. The amplificationincludes adding a common primer and repeating at least once: (1)separating the extension product from its complementary strand, (2)preferentially hybridizing the primers, and (3) extending the hybridizedprimers. The steps of the amplifying method can be repeatedindefinitely. The number of repetitions is limited by the amount ofcompetitive primers, common primers and deoxynucleotides. The extensionproducts increase exponentially. This process can be seen in FIG. 1C.This process can be used to increase the sensitivity by increasing thenumber of sequences to be detected. Thus, there is enhancement of thesequence of interest versus the background.

Alternatively, it may be advantageous to enhance the sequence ofinterest prior to an addition of the competitive oligonucleotideprimers. The method of amplifying a sequence is described in "Processfor Amplifying Nucleic Acid Sequence" U.S. Pat. No. 4,483,202 and"Process for Amplifying, Detecting, and/or Cloning Nucleic AcidSequences" U.S. Pat. No. 4,683,195, both of which are hereinincorporated by reference. Basically this method comprises the steps of:annealing an oligonucleotide primer to each strand of each differentspecific sequence; extending the primer from its 3' terminus underconditions which synthesize an extension product complementary to eachstrand, said extension product after separation from its complement,serving as a template for synthesis of the extension product of theother primer; separating the primer extension product from the templateson which they were synthesized to produce single stranded molecules; andamplifying the specific sequence by repeating the annealing, extendingand separating steps at least once.

After the amplification has occurred, the competitive oligonucleotideprimers can be added and the competitive oligonucleotide primer methodas previously described is followed.

A major distinction and advantage of the present invention over theprevious references is the competitive nature of the binding between thesubstantially complementary sequence and that containing at least onemore base mismatch. For example, the Allele specific oligonucleotide(ASO) probing method of point mutation detection uses oligonucleotidesas hybridization probes, not primers. The different ASO probes are usedin separate reactions and are not held in competition.

One of the ways that the COP procedure differs from the PCR procedure isthat PCR uses pairs of opposing primers acting at different sites toamplify specific DNA sequences while COP uses sets of primers at asingle site, to compete for binding. In the PCR procedure, more than twooligonucleotide primers may be present and these may be used to amplifydifferent sites in the same reaction vessel. Additionally, the PCR mayuse primers that are not perfectly matched to the DNA template, but areonly `substantially complementary.` However, the PCR does not utilizemixtures of primers that compete for a single binding site on thepolynucleotide template in such a way that if the most favored competingoligonucleotide primer was not present, the next most closely matchedcompeting primer would bind and prime the DNA synthesis at the samesite. Thus, it is the ability of competitive oligonucleotide primers toeach function as primers for DNA synthesis from a common site, and theprevalence of incorporation of the best-matched primer, that makes theCOP unique. The differential labeling of the individual competitiveoligonucleotide primers allows the competition event to be monitored,and thus the DNA sequence of the template can be inferred by knowing theoligonucleotide primer that is best matched.

Another major distinction from prior methods is the fact that shortprimers, about 12 mers, can be more readily used for the competitiveoligonucleotide primer assay, whereas it is usually desirable to uselonger primers for the amplification process. Because of the sensitivenature of the base mismatch assay, the longer primers, usually used foramplification, may not be as effective in a competitive assay.

The competitive oligonucleotide priming assay may function in situationswhere the precise DNA sequence to be tested is not known. A minimumrequirement is sufficient DNA sequence information to allow thesynthesis or derivation of an oligonucleotide primer that will bind toand prime DNA synthesis on the normal DNA template. Competitiveoligonucleotide primers may be used that differ from the oligonucleotideprimers that bind to the normal DNA template. Provided any one primer islabeled in a way that it may be distinguished from other competingoligonucleotide primers, then the competition between the labeled primerand other primers may be monitored by the incorporation of the labelinto the DNA extension product.

The competitive oligonucleotide priming procedure can be used for avariety of purposes. It can be used to detect known genetic sequences.One application is the detection of genetic disease. Any genetic diseasefor which the mutation(s) is known and the surrounding nucleotidesequence is known can be detected by this procedure. For example, insickle cell anemia a single base change results in the genetic disease.The mutation and surrounding sequence are known and thus the competitiveoligonucleotide primer method can be used to detect sickle cell anemia.Since the competitive oligonucleotide method is a relatively simple,quick, inexpensive and accurate assay, it should become the method ofchoice for diagnosis. For example, from the time the sample is takenuntil the result is known takes only about 6 hours.

Other diseases including thalassemia, ornithine transcarbamylasedeficiency, hypoxanthine-guanine-phosphoribosyl-transferase deficiency,and phenylketonuria can easily be detected using this process. Thismethod can be applied to cells acquired by biopsy, amniocentesis or aschorionic villi. Because the procedure measures DNA base changesdirectly, there are no limits as to the developmental time or tissuewhich must be assayed. The only requirement is that there be a knownsequence at the site that the primers will bind. This advantage can bereadily appreciated in a disease like phenylketonuria which is normallyonly expressed in the liver. The present method allows the detection ofphenylketonuria in amniotic fluid, chorionic villi or blood withoutrequiring a liver biopsy. Thus, one skilled in the art will readilyrecognize the tremendous advantages in speed, time, safety and ease ofrunning the assay of the present method.

Other applications can be readily visualized. For example, paternitytesting by testing genetic polymorphism's at any number of loci withknown sequences. With the competitive oligonucleotide primer method adifferent label could be used for each locus and thus a variety ofdifferent loci could be tested at the same time. Similarly, thecompetitive oligonucleotide primer method can be used for gene mappingand linkage analysis. Thus, even if the gene itself was not known but aclosely associated sequence with a polymorphism was known, thecompetitive oligonucleotide primer method could be used to detect thegenetic diversity at the associated sequence and to make a diagnosis.

Another area in which the method would have wide utilization is inforensic medicine. In forensic medicine, detection of genetic variationis used to provide a method to determine the origin of the sample. Thecompetitive oligonucleotide primer method provides a quick and accuratemethod to determine the sequences of a number of genetic loci from thesame sample.

The addition of extra genetic material to a genome can also be detectedby the competitive oligonucleotide primer method. For example, variousinfectious diseases can be diagnosed by the presence in clinical samplesof specific DNA sequences characteristic of the causativemicroorganisms. These include bacteria from classes such as Salmonella,Streptococcus, Staphylococcus bacilius; all fungi; all yeast; andviruses such as cytomegaloviruses, herpes simplex type I and II and HIV(Aids virus) which result in infectious disease. Again, the quick,relative, easy nature of the competitive oligonucleotide primer methodallows a ready procedure for the detection of diseases. Because of thesmall quantity of many of these microorganisms in a biological sample,it may be necessary to amplify the sequences of interest using the PCRprocedure described in U.S. Pat. Nos. 4,683,195 and 4,683,202 prior toapplying the competitive oligonucleotide primer assay of the presentinvention.

Another important use of this procedure is in the detection of neoplasmsand the monitoring of the therapy of the neoplasm. Because manyneoplasms result in the mutations of genetic sequences in the genome ofthe host or the insertion of known sequences, the competitiveoligonucleotide primer method can be used to detect these sequences.Although detection of neoplasms is important, even more useful is themonitoring of the therapy. After the institution of therapy, whether itbe by drugs surgery or radioactivity, successful neoplastic therapyresults in the disappearance of the sequence associated with thedisease. Thus, after therapy has started, samples could be taken and thecompetitive oligonucleotide primer method used to follow the course andthe effectiveness of therapy. This would provide a better prognosis forrecurrence, since very small amounts of the sequence can be detected,the test is relatively quick, and multiple samples can be monitored overtime.

In addition to the many uses of the competitive oligonucleotide primermethod in humans, there are also extensive opportunities for the use ofthe procedure in animals. In many cases, for example, horse racing andbreeding stock, in cows, pigs, dogs, cats and other animals, thecompetitive oligonucleotide primer method could be used to determine thepurity of the strain. Since determining the purity of the strain is ameasure of the sameness of the genetic sequence, and since thecompetitive oligonucleotide primer can be used to quickly and rapidlymeasure genetic sequences, it can be used as an accurate measure of thepurity of the strain in animals. As in humans, the competitiveoligonucleotide primer method can also be used for pedigree analysis anddisease screening in animals. Again, this would be important in animalhusbandry, for example, race horses, bull breeding, milk and beefbreeding, chicken breeding and pig breeding programs. In addition, sincedisease states may be accurately and quickly determined, the length ofquarantine for imported animals could be substantially diminished.

The following examples are offered by way of illustration and are notintended to limit the invention in any manner. In these examples allpercentages are by weight, if for solids, and by volume, if for liquids,and all temperatures are in degrees Celsius unless otherwise noted.

EXAMPLE 1 DETECTION OF A C TO A POINT MUTATION IN MURINE ORNITHINETRANSCARBAMYLASE COMPLEMENTARY DNA (cDNA).

An example of point mutation detection by the competitiveoligonucleotide primer method is demonstrated using cloned cDNAsequences from the murine ornithine transcarbamylase OTC gene (Veres etal. Science 237 415 (1987)). The target sequences are cloned cDNA fromnormal OTC mice, and mutant OTC cDNA from OTC deficient "sparse fur"mice, that are identical except for the substitution of an A:T base pairfor a C:G base pair at a previously determined position. Two oligomers(12-mers), complementary to either the (+) or (spf) cDNA sequences weresynthesized and are shown in Table 1 as #92 and #93, respectively. Thecomplete DNA sequence of the region surrounding the primer binding sitesis shown in FIG. 2B.

                                      TABLE 1                                     __________________________________________________________________________    OLIGONUCLEOTIDE PRIMERS                                                       No.                                                                              Sequence (5' to 3').sup.a                                                                           Length                                                                            Template                                         __________________________________________________________________________     1 CCCAGTCACGACGTT       15  M13 common                                       85 AGCTCGGTACCC          12  M13 COP                                          86 AGCTCGG(TA)AC(CG)C    12  M13 COP                                          89 AATTCGAGCTCGGTACCCGG  20  M13 COP                                          90 AATTC(GC)AGCTCGG(TA)AC(CG)CGG                                                                       20  M13 COP                                          92 CAAGTGAATGTC          12  OTC (+)                                          93 CAAGTTAATGTC          12  OTC (spf)                                        94 CTGTCCACAGAAACAGGC    18  OTC common                                       __________________________________________________________________________     .sup.a Parentheses denote mixed oligonucleotides at a single position.   

The DNA templates used in this example differ by a single DNA base pairchange. The two primers, #92 and #93, were employed along with a third,common oligonucleotide primer, #94, that was in opposite sense to thetwo competing primers (#92 and #93). The #92 and #93 primers are 12 mersin length and #94 is an 18 mer in length. Duplicate reactions wereperformed in which either the #92 or #93 oligomer was radiolabelled with³² P. In these reactions the primers #92 and #93 competed to detect thepresence of the spf mutation. Primer #94 was the common primer in thereaction.

A total of four reactions were performed: (1) normal template combinedwith radiolabelled normal primer and unlabelled mutant primer; (2)normal template combined with unlabelled normal primer and radiolabelledmutant primer; (3) mutant template combined with radiolabelled normalprimer and unlabelled mutant primer; and (4) mutant template combinedwith unlabelled normal primer and labelled mutant primer. Thus, thechemical composition of all four reactions was similar, except thateither the normal or mutant template was present with a radiolabelpresent in either the normal or mutant competing primers.

The templates were in amounts of about 500 ng each. Primer #94 waspresent at 4 μM and primers #92 and #93 were present at 2 μM each. Theprimers were radiolabelled with ³² P, at the 5'-terminus. The reactionswere carried out in about 30 μM Tris-acetate at about pH 7.9, about 10μM magnesium-acetate, about 10 μM dithiothereitol, and about 1.5 μMeach, dATP, TTP, dCTP, dGTP. The total volume of the reaction wasapproximately 100 μl. The samples were heated to about 105° for 2minutes, annealed at about 28° for 30 seconds and about 5 units of theKlenow fragment of DNA polymerase 1 from E. coli was added. The DNApolymerization continued for about 2 minutes. The heating, cooling andDNA polymerization cycle were repeated approximately 10 times. Thereaction products were analyzed by electrophoresis on a 4% NuSieve™agarose gel, followed by autoradiography.

Each reaction generated a 72 bp fragment that corresponded to the regionbetween primers #94 and either #92 or #93 (FIG. 3A). Each lane containsthe product of 10 cycles of PCR amplification using either (+) OTCcloned cDNA (lanes 1 & 2) or spf OTC cloned cDNA (lanes 3 & 4). Lanes 1and 3 contained radiolabeled primers specific to (+) OTC cDNA, lanes 2and 4 contained radiolabeled primers specific for spf OTC cDNA. The COPevent has been amplified into a 72 bp product by PCR amplification.

The presence of the 72 bp fragment in each reaction shows that efficientextension of oligonucleotide primers at each of the expected positionsoccurred and that amplification had been achieved. Autoradiographicanalysis of the agarose gel revealed preferential utilization of theperfectly matched primers at sites where the two competing primers mightbind (FIG. 3B). Thus, reactions (1) and (4) showed incorporation ofradioactivity into the reaction product and conversely, reactions (2)and (3) had no radioactivity incorporated into the reaction product. Thelevel of discrimination of the assay, that is, the degree ofpreferential utilization of the correctly matched primer over and abovethe mismatch primer, is demonstrated in FIG. 3B. Where the radioactivityis expected to be incorporated the radioactive signal is greater than100 times the case where the radioactivity is expected to be excluded.

EXAMPLE 2 COMPETITIVE OLIGONUCLEOTIDE PRIMING BY USING PRIMERS TOSEQUENCES FROM M13 mp18 FILAMENTOUS PHAGE DNA.

To further illustrate the COP principle, and to investigate theeffectiveness of the length of the competing oligomers on the COPphenomena, experiments were conducted using single stranded DNA templatederived from the filamentous phage, M13mp18. The useful feature of mp18is that it contains a region of DNA that has within its sequence therecognition sites for cleavage by a number of restriction endonucleases.When mp18 is faithfully copied by DNA synthesis, the restrictionendonuclease recognition sites are reproduced. Aberrant DNAreproduction, such as when a mismatched DNA primer is incorporated, willdestroy the restriction endonuclease recognition sites and preventcleavage by those enzymes. When two primers are simultaneously providedas primers for DNA synthesis using mp18 as a DNA template, one primerbeing perfectly complementary to the template and one primer containinga single DNA base change that destroys a restriction endonucleaserecognition site, the relative utilization of each of the two primers inthe DNA synthesis reaction may be determined by the activity of therestriction endonuclease on the synthesis product. This exampledemonstrates that perfectly matched 12-mer oligomers (A) and 20-meroligomers (B) each can be used to copy mp18 DNA and to produce asynthesis product that contains the originally present restrictionendonuclease recognition sites. Next, the 12-mers or the 20-mers weremixed with other oligonucleotide primers that were identical, except forthe presence of single DNA base substitutions that destroy restrictionendonuclease recognition sequences, and then the DNA synthesis reactionswere performed. When the primer mixture contained 12-mers, the perfectlymatched oligomer was predominantly incorporated in the DNA synthesisreaction, as revealed by the persistence of restriction endonucleaserecognition sites in the DNA synthesis products. The competition effectwas diminished where the oligomer primers were 20-mers. Thus, under theconditions used here, 12-met primers exhibit more effective COP than20-mers.

A. Competitive Oligonucleotide Primary With mp18 and 12-mers

In this example the DNA template was a single stranded DNA from thefilamentous phage M13 Mp18 (mp18). Three primers were used todemonstrate the competitive oligonucleotide priming method. A 12-merprimer, #85, which was perfectly complementary to a region of the mp18DNA template containing the restriction endonuclease recognition sitesfor RsaI and MspI, was synthesized. A 12-mer primer, #86, wassynthesized using mixed-coupling functions on an oligonucleotidesynthesizer. Primer #86 is identical to primer #85 except that at twonucleotide positions within the #86 sequence, a pair of DNA bases wasadded during synthesis. Thus, the #86 "family" was composed ofapproximately 25% AGCTCGG TAC CC, 25% AGCTCGG TAC GC, 25% AGCTCGG AACCC, and 25% AGCTCGG AAC GC. The base substitutions at the two positionswere employed to represent family members which were either (a)perfectly complementary to the mp18 template, or (b) if hybridized tothe mp18 template would generate a reaction product that no longerpossessed the correct DNA sequence for recognition and cleavage by therestriction endonucleases RsaI or MspI. A 15-mer primer, #1,complementary to the mp18 DNA, in an opposite sense to, andapproximately 75 base pairs from, the #85 and #86 binding sites was alsosynthesized. Primer #1 was the common oligonucleotide primer. Primer #1was radiolabelled ³² P at the 5'-terminus. The sequence of the primersis shown in Table 1. The DNA sequence of the region surrounding theprimer binding sites is shown in FIG. 2A.

Two reactions were performed. Each contained about 500 ng of mp18 DNAtemplate, about 4 μM radiolabelled primer #1 in about 30 μM Tris-acetateat about pH 7.4, about 50 μM sodium acetate, about 10 μM magnesiumacetate, about 10 μM dithiothreitol, and about 1.5 μM each of dATP, TTP,dCTP, dGTP the total volume was approximately 100 μl. One reactioncontained primer #85 and the other contained primer family #86. Thereaction mixtures were heated to about 105° for about 2 minutes, cooledto about 28° for about 30 seconds, about 5 units of the large fragmentof DNA polymerase 1 from E. coli were added and DNA polymerizationcarried out at about 28° for about 2 minutes. The heating, annealing andDNA polymerization were repeated 10 times. Aliquots of each reactionwere taken following 5 and 10 rounds of the amplification, and eitheranalyzed directly by gel electrophoresis and autoradiography or treatedwith a restriction endonuclease and then analyzed by gel electrophoresisand autoradiography.

FIG. 4A shows that the material which was not treated with restrictionendonuclease (un-cut) is represented by an 85 bp fragment. The 85 bpfragment is radioactive because it includes primer #1. When samples fromthe first reaction, containing primer #85, were treated with therestriction endonuclease PstI which recognizes and cleaves the DNAsequence between the binding sites for primer #1 and primer #85, then asexpected a radiolabelled 48 bp product is generated. Since the PstIrecognition sequence is between the oligonucleotide primers and shouldhave been faithfully copied during successive rounds of DNA synthesis,the PstI treated sample serves as a control.

When samples from the first reaction containing primer #85 were treatedwith the restriction endonuclease RsaI or MspI, the expectedradiolabelled 76 bp and 75 bp fragments were generated, FIG. 4A. Thepresence of the RsaI and MspI restriction endonuclease recognitionsequence indicates that primer #85 had been faithfully incorporatedduring the successive rounds of DNA synthesis.

When the products of the second reaction, containing primer family #86were analyzed in the same manner, a similar result was observed (FIG.4A). That is, restriction endonuclease PstI cleaved a 48 bp fragment,showing that the region between primers #1 and the primer family #86 wasfaithfully copied during successive rounds of DNA synthesis. However,with RsaI or MspI, complete cleavage of the 85 bp fragment was alsoobserved. This result indicates that a single member of the four memberprimer #86 oligonucleotide family was preferentially incorporated duringthe successive rounds of DNA synthesis. The failure to incorporate anyof the other three family members that contained DNA base mismatchesdemonstrates the effective competition of a perfectly matched primerwith other primers that are not perfect matches for the DNA template.

B. Competitive Oligonucleotide Priming With mp18 and 20-mers

This method using 20 met oligomers is conceptually similar to theexample above, using 12-mers.

Again a single stranded DNA from the filamentous phage M13 mp18 was usedas the template. Three primers were used to demonstrate the competitiveoligonucleotide primer method. A 20 mer primer, #89, was used which wasa perfect match for a region of mp18 DNA template containing therestriction endonuclease recognition sites for SacI, RsaI, and MspI. A20-mer primer, #90, oligonucleotide "family", was synthesized usingmixed-coupling functions on an oligonucleotide synthesizer. Primer #90is similar to primer #89 except that at three positions within the #90sequence a pair of DNA bases was added during synthesis. Thus, the #90oligonucleotide family has eight members, 1 containing DNA sequencesrecognized by all 3 restriction endonucleases, 3 containing combinationsof 2 sites, 3 containing 1 site and 1 having no restriction sites.Furthermore, each site is equally represented by a perfect match for themp18 DNA template. The 15-mer primer, #1, was employed as the commonprimer. The sequence of the primers is shown in Table 1. The DNAsequence surrounding the primer binding sites is shown in FIG. 2A. Theconditions for each reaction were the same in those used above in thecompetitive oligonucleotide priming of mp18 with 12-mers.

When samples from the reactions containing primer #89 were takenfollowing 10 cycles of DNA synthesis and treated with the restrictionendonucleases PstI, SacI, RsaI or MspI, then a 91 bp radiolabelledfragment was observed that could be reduced to 81, 76, or 75 bp's by therestriction endonucleases, respectively. (FIG. 4B). This indicated thatboth the region between the primers #1 and 89 and the DNA sequencesoverlapped by primer number 89 were faithfully copied during therepeated rounds of DNA synthesis.

In contrast, some of the products of the reaction containing theoligonucleotide primer family #90 were refractory to cleavage by therestriction endonucleases SacI, RsaI and MspI (FIG. 4B). The restrictionendonuclease PstI that recognizes DNA sequences between theoligonucleotide priming sites, efficiently cleaved the reaction product.The failure to cleave was therefore due to the incorporation of themismatched oligonucleotides from the #90 oligonucleotide family. Thus,while the experiment with mp18 DNA and 12 mers, Example 2(A) above,illustrates that, under these reaction conditions, 12 mers may exhibiteffective competition for a unique DNA priming site, longer (20 mer)oligomers may exhibit less effective competitive oligonucleotidepriming.

EXAMPLE 3 β^(s) -GLOBIN (SICKLE CELL ALLELE)

The mutation in humans causing sickle cell anemia is also detectable bycompetitive oligonucleotide priming. The normal DNA sequence (β⁺) of thehuman β-globin gene in the region of the β-sickle cell allele is:##STR1## And the site of the single DNA base change that gives rise tothe β^(s) (Sickle Cell) hemoglobinopathy is indicated by an arrow. Thebase change leading to the β^(s) genotype is A→T. Thus, the followingprimers can be constructed and used in the competitive oligonucleotideprimer assay:

(1) 5'-CTC.CTG.AGG.AGA.-3' (12-mer -β⁺ specific)

(2) 5'-CTC.CTG.TGG.AGA.-3' (12-mer -β^(s) specific)

Primers (1) and (2) can be differentially labelled and then usedsimultaneously in a COP reaction with either cloned β⁺ -globin or β^(s)-globin sequences.

The successfully competing primer is then identified using a thirdprimer to amplify the extension product of the competitive primer whichsuccessfully bound the DNA:

(3) 5'-CGT.TCA.CCT.TGC.CCC.ACA.GG-3' Primer 3 will prime DNA synthesisin an opposite direction to primers 1 or 2. When this assay is performedusing the primer trio 1, 2 and 3, then a 47 bp fragment is produced andwould indicate the true DNA sequence of the DNA template.

If the starting material to assay was a complex DNA mixture, e.g. humangenomic DNA, then two primers may be first used to amplify the sitecontaining the β^(s) mutant allele.

For example, the oligonucleotides

5'-TGG.TCT.CCT.TAA.ACC.TGT.CTT.G-3'

5'-ACA.CAA.CTG.TGT.TCA.CTA.G-3'

amplify a 167 bp segment of the human β-globin gene, containing DNAcomplementary to primers 1, 2 and 3 described herein.

Following amplification of the region containing the β^(s) mutation, thecompetitive oligonucleotide primer assay can be performed as describedabove. In each case, the oligomers corresponding to either the B⁺ orβ^(s) alleles would be labeled, so as to be distinguished from the otheroligomers. The detection of the unique label is the end point of theassay.

EXAMPLE 4 α₁ -ANTITRYPSIN Z ALLELE; S ALLELE.

The mutations in humans leading to deficiency of α₁ -antitrypsin is alsodetectable by competitive oligonucleotide priming. The normal human DNAsequence (M) in the α₁ -antitrypsin gene, surrounding the sitecontaining the α₁ -Z allele is: ##STR2## and the mutation shown at thearrow (G→A) gives rise to the mutant Z allele.

Similarly, the normal human DNA sequence (M) in the α₁ antitrypsin gene,surrounding the site containing the α₁ -S allele is: ##STR3## and themutation shown (A→T) gives rise to the S allele.

Thus, primers specific for the discrimination of the M/Z allele pair orthe M/S allele pair can be constructed:

(A) M/Z

(1) 5'-ATC.GAC.GAG.AAA.-3' (M)

(2) 5'-ATC.GAC.AAG.AAA.-3' (Z)

(B) M/S

(3) 5'-ACC.TGG.AAA.ATG.-3' (M)

(4) 5 '-ACC.TGG.TAA.ATG.-3' (S)

Primers (1) and (2), and (3) and (4) can be differentially labeled andused in the competitive oligonucleotide primer assay to distinguishcloned normal (M) or mutant (Z or S) α₁ -antitrypsin DNA sequences. Theextension products of the successfully competing primers can bedetected, after amplification, through the use of common primers.

For example, primers (1) and (2) plus primer (5)(5'-CAG.CCA.GCT.TCA.GTC.CCT.TTC-3') will together produce a fragment of81 bp in the reaction. Primers (3) and (4) plus primer (6)(5'-GGG.AAT.CAC.CTT.CTG.TCT.TC-3') will produce a fragment of 70 bp.

If the starting material includes samples of human genomic DNA, then theprimer sets, 5'-ACG.TGG.AGT.GAC.GAT.GCT.CTT.CCC-3' and5'-GTG.GGA.TTC.ACC.ACT.TTT.CCC-3', that flank but do not include themutation site for the Z allele can be employed to preamplify a 450 bpfragment of the α₁ -antitrypsin gene containing the Z allele. Primersets 5'-GAA.GTC.AAG.GAC.ACC.GAG.GAA-3' and5'-AGC.CCT.CTG.GCC.AGT.CCT.AGT.G-3' which flank but do not include themutation site for the S allele may be employed to preamplify a 340 bpregion of the α₁ -antitrypsin gene containing the S allele.

The amplified mutation sites could then be utilized as starting materialfor the COP analysis using the competitive oligonucleotides and theiropposing common primers. For instance, primers (1) and (2) compete tobind to the site of the Z allele within the amplified 450 bp fragment.The extension product of the successfully competing primer, either (1)or (2), can then be detected after amplification through the use of thecommon primer (5). Similarly, primers (3) and (4) compete to bind to thesite of the S allele within the 340 bp fragment and the extensionproduct of the successfully competing primer, either (3) or (4),detected after amplification through the use of the common primer (6).

One skilled in the art will readily appreciate the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as, those inherent therein. The methods, proceduresand techniques described herein are presently representative of thepreferred embodiments, are intended to be exemplary, and are notintended as limitations on the scope. Changes therein and other useswill occur to those skilled in the art which are encompassed within thespirit of the invention or defined by the scope of the appended claims.

What is claimed is:
 1. A method for detecting the presence or absence ofan oligonucleotide having a specific known nucleic acid sequence, ordistinguishing between oligonucleotides having different sequences,comprising the steps of:adding oligonucleotide primers to a sample ofnucleic acid or mixture of nucleic acids, wherein said oligonucleotideprimers include at least two competitive primers, a first primer beingsubstantially complementary to a portion of the specific known sequenceoligonucleotide and at least one second primer also being substantiallycomplementary to said portion but having at least one additional basemismatch with said portion of the specific known sequenceoligonucleotide, relative to the first primer, whereby said first andsecond primers compete for said portion of the specific known sequence,and wherein the oligonucleotide primers are in molar excess to saidsample of nucleic acid; preferentially hybridizing the first primer tothe specific known sequence oligonucleotide under competitiveconditions, wherein each competitive primer will hybridize to the knownsequence oligonucleotide in the absence of other competitive primers,but wherein the more complementary first primer preferentiallyhybridizes to the exclusion of other less complementary second primerspresent; extending the preferentially hybridized first primer from its3' terminus to synthesize an extension product complementary to thespecific known sequence oligonucleotide to which the preferentiallyhybridized primer is hybridized; and detecting the presence or absenceof said extension product as a measure of the presence or absence ofsaid specific known sequence oligonucleotide.
 2. The method of claim 1,wherein said competitive oligonucleotide primers are about 8-24nucleotides long.
 3. The method of claim 1, wherein at least one of saidcompetitive oligonucleotides primers is labeled and said extensionproduct is identified by determining the presence or absence of saidlabel in said extension product.
 4. The method of claim 3, wherein saidlabel is selected from the group consisting of radioisotopes,fluorescers, chemiluminescers, enzymes and antibodies.
 5. The method ofclaim 1, comprising the further steps of:adding a common oligonucleotideprimer prior to said identifying step, wherein said common primerhybridizes to the strand complementary to the strand that thecompetitive primers hybridize to and at a site distant from said portionto which said competitive primers hybridize; extending said commonprimer at a time when said preferentially hybridized primer is extended,and wherein the extension product from said common primer and saidpreferentially hybridized primer are complementary to each other;separating said extension product from its complementary strand; andrepeating said preferentially hybridizing and said extending steps. 6.The method of claim 5, comprising the further step of repeating at leastonce, said separating, preferentially hybridizing and extending steps.7. A competitive oligonucleotide primer method for detecting thepresence or absence of an oligonucleotide having a specific knownnucleic acid sequence in a sample containing a mixture of separatecomplementary nucleic acid strands, or distinguishing between at leasttwo oligonucleotides having different sequences in the sample,comprising the steps of:adding a common primer and oligonucleotideprimers to a sample of nucleic acid or mixture of nucleic acids, whereinsaid oligonucleotide primers include at least two competitive primers, afirst competitive oligonucleotide primer being substantiallycomplementary to a portion of the specific known sequenceoligonucleotide and at least one second primer also being substantiallycomplementary to said portion but having at least one base lesscomplementarity with the specific known sequence oligonucleotide,relative to the fist primer, whereby said first and second primerscompete for said portion of the specific known sequence, wherein saidoligonucleotide primers are in molar excess to said sample of nucleicacid, and wherein said common primer hybridizes to the strandcomplementary to the strand that the competitive primers hybridize toand at a site distant from said portion to which said competitiveprimers hybridize; annealing said competitive oligonucleotide primer andsaid common primer to said separate complementary strands underconditions in which said common primer anneals to one of thecomplementary strands and said competitive oligonucleotide primeranneals to the other said complementary strand containing the specificknown sequence, thereby forming annealed primers, wherein eachcompetitive oligonucleotide primer will hybridize to the specific knownsequence oligonucleotide in the absence of other competitiveoligonucleotide primers, but wherein the more complementary primerpreferentially hybridizes to the exclusion of other less complementaryprimers present, and wherein said first competitive oligonucleotide willbind preferentially to the known sequence over said second competitiveoligonucleotide; extending said annealed primers from their 3' terminusto synthesize extension products complementary to the complementarystrands annealed to said annealed primers; separating said extensionproducts from said complementary strands to produce single-strandedmolecules, said extension product of one said complementary strand,after separation from its complement, serving as a template for thesynthesis of said extension product of an other of said complementarystrands; amplifying said single-stranded molecules comprising saidspecific known sequence by repeating, at least once, said annealing,extending and separating steps thereby forming an amplified extensionproduct; and detecting the presence or absence of said amplifiedextension product as a measure of the presence or absence of saidspecific known sequence oligonucleotide.
 8. The method of claim 7,wherein at least one of said competitive oligonucleotide primers islabeled with a label, wherein if more than one competitiveoligonucleotide primer is labeled, each competitive oligonucleotideprimer has a different label attached depending on the sequence of theoligonucleotide primer, and wherein said label is selected from thegroup consisting of radioisotopes, fluorescers, chemiluminescers,enzymes and antibodies.
 9. The method of claim 8, wherein saididentifying step includes detecting the presence or absence of saidlabeled primer in the amplified extension product.
 10. The method ofclaim 8, wherein said common oligonucleotide primer is bound to a solidsupport, and wherein said amplified extension product is allowed tohybridize with said common oligonucleotide primer and thus becomesattached to said solid support by virtue of said hybridization, andwherein said amplified extension product is identified by measuring thepresence or absence of said label attached to said solid support. 11.The method of claim 7, wherein said specific nucleic acid sequencecontains at least one mutation that causes a genetic disease.
 12. Acompetitive oligonucleotide primer method for detecting the presence orabsence of a plurality of oligonucleotides having specific known nucleicacid sequences in a sample containing a mixture of nucleic acid strands,comprising the steps of:annealing a specific oligonucleotide primer toeach specific known sequence oligonucleotide, thereby forming annealedprimers; extending each of said annealed primers from its 3' terminus tosynthesize extension products complementary to said specific knownsequence annealed to the primers; separating said extension productsfrom said specific known sequence oligonucleotides on which they weresynthesized to produce single-stranded molecules, said extensionproduct, after separation serving as a template for synthesis ofadditional extension product of said specific oligonucleotide primers;amplifying said single-stranded molecules comprising said specific knownsequence by repeating, at least once, said annealing, extending andseparating steps; adding oligonucleotide primers to said sample, whereinsaid oligonucleotide primers include at least two competitive primersfor each specific known sequence oligonucleotide, a first primer beingsubstantially complementary to a portion of said specific known sequenceand at least one second primer also being substantially complementary tosaid portion but having at least one base less complementarity with saidportion of the specific known sequence oligonucleotide, relative to thefirst primer, whereby said first and second primers compete for saidportion of the specific known sequence, and wherein the oligonucleotideprimers are in molar excess to said sample of nucleic acid;preferentially hybridizing said first primer to said specific knownsequence oligonucleotide under competitive conditions thereby formingpreferentially hybridized primers, and wherein said first competitiveoligonucleotide will bind preferentially to the known sequence over saidsecond competitive oligonucleotide; extending said preferentiallyhybridized primer from its 3' terminus to synthesize a competitiveextension product complementary to said strand to which it ishybridized; and detecting the presence or absence of said amplifiedextension product as a measure of the presence or absence of saidspecific known sequence oligonucleotide.
 13. The method of claim 12,wherein said competitive oligonucleotide primers are about 8-24nucleotides long.
 14. The method of claim 12, wherein at least one ofsaid competitive oligonucleotide primers, having a label, is extendedand said competitive extension product is identified by determining thepresence or absence of said label in said competitive extension product.15. The method of claim 14, wherein, the label is selected from thegroup consisting of radioisotopes, fluorescers, chemiluminescers,enzymes and antibodies.
 16. The method of claim 12, further comprisingthe steps of:adding a common oligonucleotide primer prior to saididentifying step, wherein said common primer hybridizes to the strandcomplementary to the strand that the competitive primers hybridize toand at a site distant from said portion to which said competitiveprimers hybridize; separating said competitive extension product fromits complementary strand; and repeating said preferentially hybridizingand said extending steps.
 17. The method of claim 16, comprising thefurther step of repeating at least once said separating, preferentiallyhybridizing and extending steps.
 18. A competitive oligonucleotideprimer method of detecting a genetic condition which results from atleast one mutation in a specific known nucleic acid sequence, comprisingthe steps of:adding oligonucleotide primers to a sample of nucleic acidor a mixture of nucleic acids, wherein said oligonucleotide primersinclude at least two competitive primers, a first primer beingsubstantially complementary to the normal genetic sequence and at leastone second primer being substantially complementary to the mutatedgenetic sequence, wherein the competitive primers compete with eachother for hybridization to said sample of nucleic acid, and wherein thecompetitive oligonucleotide primers are in molar excess to said sampleof nucleic acid; preferentially hybridizing said first and secondprimers to the sample of nucleic acid under competitive conditionsthereby forming preferentially hybridized primers; extending saidpreferentially hybridized primers from their 3' terminus to synthesizeextension products complementary to said strands to which thepreferentially hybridized primers are hybridized; and detecting thepresence or absence of said extension product as a measure of thepresence of absence of said specific known oligonucleotide.
 19. Themethod of claim 18, wherein at least one of said competitiveoligonucleotide primers is labeled with a label, said label beingselected from the group consisting of radioisotopes, fluorescers,chemiluminescers, enzymes and antibodies.
 20. The method of claim 18,wherein the competitive oligonucleotide primers have a different labelattached depending on the sequence of said competitive oligonucleotideprimer, said label being selected from the group consisting ofradioisotopes, fluorescers, chemiluminescers, enzymes and antibodies.21. The method of claim 18, comprising the further steps of:dividingsaid sample into a plurality of portions prior to adding saidcompetitive oligonucleotide primers; adding competitive oligonucleotideprimers to each of said portions, wherein each portion will differ fromthe other said portions because a different primer in the set of saidcompetitive primers has said label; and measuring the presence orabsence of said label in said extension products in each of saidportion.